Apparatus for retaining magnetic particles within a flow-through cell

ABSTRACT

An apparatus for retaining magnetic particles within a segment of a flow-through cell during flow of a fluid through the cell comprises (a) optionally, an electrical current source; (b) an electromagnet having a winding connected to the current source and an air gap between at least one pair of poles each of which has a corrugated outer surface and (c) a flow-through cell which is configured and dimensioned to receive an amount of magnetic particles to be retained within the flow-through cell and to allow flow of a liquid through the flow-through cell. The liquid carries molecules or particles to be captured by means of the magnetic particles. A portion of the flow-through cell is inserted in air gap.

FIELD OF THE INVENTION

The invention concerns an apparatus and a method for retaining magneticparticles within a segment of a flow-through cell during flow of a fluidthrough the cell.

The invention further concerns an apparatus and a method of the abovekind which is in addition adapted for manipulating magnetic particlesretained within a segment of a flow-through cell during flow of a fluidthrough the cell.

The invention concerns in particular an apparatus and a method of theabove mentioned kinds wherein the magnetic particles are used forcapturing target molecules or target particles suspended in and carriedby a fluid flowing through a flow-through cell, as is done for instancein clinical chemistry assays for medical diagnostic purposes. Theinvention further concerns use of an apparatus and a method of the abovementioned kinds in the field of life sciences and in particular forin-vitro diagnostics.

BACKGROUND OF THE INVENTION

Magnetic separation and purification processes using magnetic particlesas a solid extraction phase are widely used e.g. in clinical chemistryassays for medical diagnostic purposes, wherein target molecules ortarget particles are bound on suitable magnetic particles and labeledwith a specific receptor, and these method steps are followed by a stepwherein the magnetic particles carrying target particles bound on themare separated from the liquid where they were originally suspended bymeans of a high magnetic field gradient.

Within the scope of this description the terms target molecules orparticles are used to designate in particular any biological componentssuch as cells, cell components, bacteria, viruses, toxins, nucleicacids, hormones, proteins and any other complex molecules or thecombination of thereof.

The magnetic particles used are e.g. paramagnetic or superparamagneticparticles with dimension ranging from nanometric to micrometric scales,for instance magnetic particles of the types mentioned in thepublication of B. Sinclair, “To bead or not to bead,” The Scientist,12[13]:16-9, Jun. 22, 1998.

The term specific receptor is used herein to designate any substancewhich permits to realize a specific binding affinity for a given targetmolecule, for instance the antibody-antigen affinity (see e.g. U.S. Pat.No. 4,233,169) or glass affinity to nucleic acids in a salt medium (seee.g. U.S. Pat. No. 6,255,477.

Several systems using magnetic separation and purification process havebeen developed during the two last decades and have led to a largevariety of commercially available apparatus which are miniaturized andautomated to some extent, but there has been relatively little progressin the development of the means used in those apparatuses for handlingthe magnetic particles. Basically the process comprises the step ofmixing of a liquid sample containing the target molecules or particleswith magnetic particles within a reservoir in order that the bindingreaction takes place and this step is followed by a separation step ofthe complexes magnetic particle/target particle from the liquid by meansof a permanent magnet or an electromagnet. Since this separation step isusually carried out with the liquid at rest, this step is known asstatic separation process. In some systems additional steps required forhandling of the liquids involved (liquid sample, liquid reagent, liquidsample-reagent mixtures) are carried out by pipetting means.

A flow-through system for carrying out the separation of the magneticparticles, a so called dynamic separation system, is more advantageousthan a static separation system, in particular because it makes possibleto effect separation of magnetic particles and steps involving liquidprocessing with more simple means and with more flexibility.

However, only few magnetic separation systems are known and they haveserious drawbacks. In most of them the magnetic particles retained builda cluster deposited on the inner wall of a flow-through cell and forthis reason the perfusion of the target molecules is inefficient.

According to U.S. Pat. No. 6,159,378 this drawback can be partiallyovercome by inserting in the flow path of the liquid carrying the targetmolecules or target particles a filter structure made magnetic fluxconducting material, and by applying a magnetic field to that filterstructure. A serious drawback of this approach is that the filterstructure is a source of contamination or cross-contamination problems.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an apparatus and amethod by which the magnetic particles retained are homogeneouslydistributed over the cross-section of the flow-through cell, so thatliquid flowing through the flow-through cell flows through the retainedparticles and a maximum of the surfaces of the particles is contacted bythe liquid during that flow, thereby enabling an efficient capture ofthe target molecules or target particles.

In another embodiment, the present invention provides an apparatus and amethod in which the magnetic particles which serve for capturing targetparticles carried by a liquid sample which flows through a flow-throughcell are so retained therein that they are homogeneously distributed inthe interior of the flow-through cell, thereby enabling a highlyeffective perfusion of the particles retained, because the liquid samplecarrying the target particles flows through a kind of filter structurebuilt by the magnetic particles themselves, and this effect is obtainedwithout having within the flow-through cell any component which might bea possible source of contamination or cross-contamination.

In another embodiment, the present invention provides an apparatus and amethod such that usual steps like washing or eluting of the magneticparticles and of the target particles bound on them can also be effectedwith the same apparatus and this leads to a very rapid automatedprocessing of sample liquids and to a corresponding reduction of thecost of such processing.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject invention will now be described in terms of its preferredembodiments with reference to the accompanying drawings. Theseembodiments are set forth to aid the understanding of the invention, butare not to be construed as limiting.

FIG. 1 shows a schematic front view of an apparatus according to theinvention and also related axis Y and Z.

FIG. 2 shows an enlarged side view of zone 20 in FIG. 1 and also relatedaxis X and Y.

FIG. 3 an enlarged side view similar to FIG. 2 and showing the spatialdistribution of magnetic particles retained within a segment of aflow-through cell.

FIG. 4 shows an enlarged side view similar to FIG. 2 wherein it isschematically depicted that the pole tips of 21 and 22 generate a highmagnetic field gradient over the entire cross-section of air gap 23.

FIG. 5 is a diagram showing the spatial variation of the magnetic fieldintensity created with pole tips 21, 22 in FIG. 1 along the length axis(X-axis) at the middle of air gap 23.

FIG. 6 shows a perspective view of electromagnet 13 as seen in FIG. 1.

FIG. 7 shows an exploded view of the components of the electromagnetrepresented in FIG. 6.

FIG. 8 shows a cross-sectional view of the distribution of the magneticparticles in flow-through cell 18 when they are under gravity forcealone, that is with no magnetic field applied, or when a static magneticfield is applied and the density of magnetic particles is lower that acertain limit value.

FIG. 9 shows a cross-sectional view of the distribution of the magneticparticles retained in flow-through cell 18 when an alternating magneticfield is applied according to the invention and even when a relativelylow density of magnetic particles is used.

FIG. 10 shows a diagram (flow in milliliter per minute) vs. magneticfield (in Tesla) illustrating the retention capability of an apparatusoperating with an alternating magnetic field of 2 cycles per second anda flow-through cell 18 having an internal diameter of 1.5 millimeter.

FIG. 11 shows a perspective view of a two-dimensional corrugated patternof the pole surfaces suitable for generating a magnetic gradient havinga three dimensional distribution.

FIG. 12 schematically illustrates use of an apparatus wherein the polesof the electromagnet have outer surfaces having the shape shown in FIG.11 and a plurality of flow-through cells are inserted in the air gapbetween those outer surfaces.

FIG. 13 schematically illustrates use of an apparatus wherein the polesof the electromagnet have outer surfaces having the shape shown in FIG.11 and a plurality of flow-through cells fluidically connected in seriesis inserted in the air gap between those outer surfaces.

FIG. 14 shows a perspective view of a quadrupole configuration of poleshaving corrugated surfaces suitable for generating a magnetic gradienthaving a symmetric distribution enabling a more homogeneous distributionof magnetic particles.

FIG. 15 shows a cross-sectional view of the quadrupole configuration ofpoles shown by FIG. 14.

FIG. 16 shows a schematic view of a fourth example of an apparatusaccording to the invention.

FIG. 17 shows a perspective view of the apparatus shown by FIG. 16.

FIG. 18 shows a perspective exploded view of components of a fifthexample of an apparatus according to the invention.

FIG. 19 shows a top view of layer 101 in FIG. 18 and of theferromagnetic material sheets 107 and 108 inserted in cavities 105 and106 of layer 101.

FIG. 20 shows a cross-sectional view of the apparatus shown by FIGS. 18and 19 further including an electromagnet 121.

DETAILED DESCRIPTION OF PREFERRED EXAMPLES First Apparatus Example

A first example of an apparatus according to the invention is describedhereinafter with reference to FIGS. 1 to 10. FIG. 1 shows a schematicfront view of an apparatus according to the invention and also relatedaxis Y and Z. FIG. 2 shows an enlarged side view of zone 20 in FIG. 1and also related axis X and Y.

As shown by FIG. 1, an apparatus according to the invention comprises:

-   -   (a) optionally, an electrical current source 12;    -   (b) an electromagnet 13 comprising a winding 14 connected to the        current source 12, and    -   (c) a flow-through cell 18 which is configured and dimensioned        to receive an amount of magnetic particles to be retained within        a segment of the flow-through cell and to allow flow of a liquid        through the flow-through cell.

In a preferred embodiment the electric current source 12 is a sourceadapted to provide a current which is variable with time, e.g. analternating current source adapted to supply a current having aselectable frequency comprised between 0.001 cycle per second and 100kilocycles per second.

In another embodiment electric current source 12 is a switchable DCcurrent source.

In another embodiment electric current source 12 is a DC current source.

When a DC current is applied to winding 14, the magnetic particlesmigrate to the region were the magnetic field is highest following thespatial variation of the magnetic field, and this effect forms aperiodic distribution of chains of magnetic particles located atdifferent segments 41 along the channel of the flow-through cell asshown by FIG. 3. However, since the magnetic field is highest near themagnetic poles, the magnetic particles will be concentrated at the wallsof the flow-through channel and near the magnetic poles. Moreoverlateral observations of the tube cross-section show that the magneticparticles do not cover the whole cross section due to the deposition ofthe magnetic particles under gravity force as shown by FIG. 8. With suchmagnetic particle aggregations, a very low surface of the magneticparticles will be in contact with only a limited volume of the fluidflow. By increasing the magnetic particles density, one cansystematically cover more cross-section surface of the flow-channel andthus increase the fluid flow volume which is in contact with themagnetic particles surface. Nevertheless, in this case the surface ofthe magnetic particles in contact with the fluid flow is still very lowcompared with their total volume and one could have a serious problem ofbackpressure and even the absence of a flow. This problem is overcome byapplying an AC current to winding 14 in order to induce a local dynamicbehavior of the magnetic particles. This dynamic behavior is dictatedessentially by the fact that the minimum energy of a magnetic particlein an applied magnetic field is reached when the dipolar magnetic momentvector of this particle is parallel to the applied magnetic field. Underthe influence of a magnetic field the magnetic particles tend to formchains which have particular dynamic behaviors at different frequenciesof the magnetic field applied. At low frequencies, the magneticparticles form chain structures that behave like a dipole, which isreversed by a change of the magnetic field polarity. At high frequenciesthe magnetic particles have a vortex rotational dynamic. Such arotational dynamic seems to be useful to provide a more efficienthomogeneous distribution of the magnetic particles over thecross-section of the flow channel as shown by FIG. 9, even when arelatively low density of the magnetic particles is used. Moreover, thisdynamic behavior is particularly interesting since it permit to have amore efficient interaction between the magnetic particles and the targetparticles carried by a liquid that flows through the flow-through cell.

Electromagnet 13 has at least one pair of poles 21, 22 separated by anair gap 23 which is much smaller than the overall dimensions of theelectromagnet. Electromagnet 13 comprises yoke parts 15, 16, 17, poleend parts 21, 22 and a winding 14 connected to electrical current source12.

Air gap 23 lies between outer surfaces 24, 25 of the ends of the poles.Each of these outer surfaces comprises the outer surfaces of at leasttwo cavities 31, 33 respectively 34, 36 and of a tapered pole end part32 respectively 35 which separates the two cavities 31, 33 respectively34, 36 from each other. Air gap 23 has an average depth which liesbetween 0.1 and 10 millimeters.

Cavities 31, 33 and the tapered end part 32 of one of the poles 21 arearranged substantially opposite to and symmetrically with respect to thecorresponding cavities 34, 36 and tapered end part 35 of the other pole22 of the pair of poles. The depth of air gap 23 thereby varies at leastalong a first direction, e.g. the X-direction. This depth is measuredalong a second direction, e.g. the Y-direction, which is normal to thefirst direction. Air gap 23 has at least a first symmetry axis whichextends along the first direction, i.e. the X-direction.

As can be appreciated from FIG. 2, in a preferred embodiment each oftapered pole end parts 32, 35 has a sharp edge. In another embodimentshown by FIG. 3, the cross-section of the outer surface 24 a, 25 a ofthe pole ends 21 a, 22 a has an undulated or sawtooth shape.

Each of tapered pole end parts 32, 35 has in general a three-dimensionalshape and the cavities 31, 33 respectively 34, 36 and tapered pole endparts 32 respectively 35 form a corrugated surface. In preferredembodiments this corrugated surface has a thickness comprised between0.1 and 10 millimeters.

Each of above mentioned tapered pole end parts, e.g. pole parts 21, 22,is made of a ferromagnetic material and preferably of a ferrite.Cavities 31, 33 respectively 34, 36 are made by a suitable process, e.g.by micro powder blasting.

As schematically shown by FIG. 4, pole tips of 21 and 22 generate a highmagnetic field gradient over the entire cross-section of air gap 23. InFIG. 4 dashed lines 26 represent magnetic field lines.

FIG. 5 shows a diagram of a representative spatial variation of themagnetic field intensity created with pole tips 21, 22 in FIG. 1 alongthe length axis (X-axis) at the middle of air gap 23 and for a currentdensity of 2 A/square millimeter. In this diagram the intensity of themagnetic field is expressed in Ampere/meter and the position along theX-axis is indicated by a length expressed in millimeters. As can beappreciated from FIG. 5, the magnetic field and the magnetic fieldgradient have simple and well defined periodic forms which arecontrolled by the electrical and geometrical characteristics ofelectromagnet 13, and in particular by the shape of the pole tips.

When flow-through cell 18 is used according to the invention, the liquidwhich flows through it carries target molecules or target particles tobe captured by means of magnetic particles retained within theflow-through cell.

In another embodiment, flow-through cell 18 is made of a material whichhas no magnetic screening effect on a magnetic field generated byelectromagnet 13.

A portion of the flow-through cell 18 is inserted in the air gap 23 insuch a way that at least one area of the outer surface of each of thetapered pole parts 32, 35 is in contact with or is at least very closeto the outer surface of a wall 19 of the flow-through cell and thelength axis of the flow-through cell portion extends along the firstdirection, i.e. the X-direction.

The magnetic particles used are of the kind used for capturing targetmolecules or target particles carried by a liquid. The size of themagnetic particles lies in the nanometer or micrometer range.

In another embodiment, magnetic particles suitable for use within thescope of the invention have e.g. the following characteristics:

-   -   a diameter of 2 to 5 micrometer    -   a magnetic force of approximately 0.5 Newton per kilogram.

Properties of the magnetic particles suitable for use within the scopeof the invention are described in particular in the following patentspecifications: EP 1154443, EP 1144620, U.S. Pat. No. 6,255,477.

FIG. 6 shows a perspective view of electromagnet 13 in FIG. 1. FIG. 7shows an exploded view of the components of the electromagnetrepresented in FIG. 6.

In the embodiment shown by FIGS. 6 and 7, cavities 31, 33 respectively34, 36 are grooves or channels parallel to each other. The length axisof each of such grooves or channels extends along a third direction,e.g. the Z-direction, which is normal to a plane defined by a first axisin the first direction, i.e. the X-direction, and a second axis in thesecond direction, i.e. the Y-direction.

The grooves of channels have a cross-section which has e.g. the shape ofa half circle as shown by FIG. 2 or an undulated or sawtooth shape asshown by FIG. 3.

Second Apparatus Example

A second example of an apparatus according to the invention is shown byFIG. 11. This embodiment has all basic features described above for thefirst apparatus example, but outer surfaces of the electromagnet poles51, 52 which define an air gap 53 are corrugated surfaces 54, 55, eachof which comprise tapered pole end parts which are arranged in a matrixarray. In this second embodiment the at least two cavities(corresponding to cavities 31, 33 respectively 34, 36 in FIG. 2) and thetapered pole end parts (corresponding to 32 respectively 35 in FIG. 2)are also opposite to and symmetrical with respect to each other and areformed by the intersection of

-   -   a first set of grooves or channels parallel to each other, the        length axis of each of those grooves or channels extending along        a third direction, e.g. the Z-direction, which is normal to a        plane defined by a first axis in the first direction, i.e. the        X-direction, and a second axis in the second direction, i.e. the        Y-direction, with    -   a second set of grooves or channels parallel to each other, the        length axis of each of the grooves or channels extending along        the first direction (X-direction).

As shown by FIG. 11, each of the grooves or channels of the first set ofgrooves or channels, and also of the second set of grooves or channels,has e.g. a cross-section with the shape of a half circle. In a variantof this embodiment the latter cross-section has e.g. a wave-like orsawtooth shape.

As shown by FIG. 11, each of the tapered pole end parts (correspondingto tapered pole end parts 21, 22 in FIG. 2) has a flat outer surfacefacing the air gap (corresponding to air gap 23 in FIG. 2). In a variantof this embodiment, each of the tapered pole end parts ends in a ridge.

In the embodiment represented by FIG. 1 1 one or more flow-through cells(not represented in FIG. 11) may be inserted into gap 53.

Examples of two possible uses of the embodiment represented by FIG. 11are schematically represented in FIGS. 12 and 13.

In the example shown by FIG. 12 a plurality of flow-through cells 61,62, 63, 64 having each an inlet and an outlet are inserted in air gap 53between outer surfaces 54 and 55 in FIG. 11. Several liquid samples,which may be different ones, can thus flow through flow-through cells61, 62, 63, 64, e.g. in the sense indicated by arrows in FIG. 12. InFIG. 12 the pole tips are represented by rectangles like 71, 72, 73, 74located close to flow-through cell 61.

In the example shown by FIG. 13 a plurality of flow-through cellsfluidically connected in series or a plurality of segments of a singleflow-through cell 65 having the meander shape shown in FIG. 13 areinserted in air gap 53 between outer surfaces 54 and 55 in FIG. 11. Thisflow-through cell arrangement 65 has an inlet and an outlet and a liquidsample can flow therethrough in the sense indicated by arrows in FIG.13.

In FIG. 13 the pole tips are also represented by rectangles like 71, 72,73, 74 located close to flow-through cell 65.

In the embodiments represented in FIGS. 12 and 13 each of the rectangles71, 72, 73, 74 representing a pole tip surface has a width H and a depthh, and the distance separating successive pole tips in the same row orcolumn of the matrix array of pole tips is designated by the letter l.

In the case of an embodiment comprising a single row of pole tips, thedepth h may be chosen tp be equal to the width of the channel defined bythe flow-through cell, the width H can e.g. lie in a range going from0.1 to 10 millimeter and the dimension l can be defined e.g. by l=2*H, auniform distribution of the magnetic particles is obtainable e.g. in aflow-through cell having a diameter of 1 millimeter and a length of 16millimeter using 8 pole tips each of which has a dimension H=0.1millimeter, when a mass of about 2 milligrams of magnetic particles areused, an alternating magnetic field is used which has a frequency withina range going from 1 to 15 cycles per second, and the magnetic particlesused have e.g. the following characteristics: a diameter of 2 to 5micrometer and a magnetic force of approximately 0.5 Newton perkilogram.

An example of use of an embodiment comprising a single row of pole tipsof the type just mentioned above is the use of such an embodiment forthe capture of λ-DNA. In this example the parameters involved have e.g.the following values:

The depth h may be equal to the width of the channel defined by theflow-through cell

-   -   H=1 millimeter    -   Mass of magnetic particles used: between 2 and 5 milligram

Characteristics of the magnetic particles used:

-   -   a diameter of 2 to 5 micrometer, and    -   a magnetic force of approximately 0.5 Newton per kilogram.

Diameter of the channel

-   -   of the flow-through cell=1.5 millimeter

Length of the channel

-   -   of the flow-through cell=16 millimeter    -   Number of pole tips=6    -   Mass of DNA used=2 microgram

Frequency of alternating magnetic field applied in a range going from 1to 15 cycles per second.

The test results obtained with the above defined operating conditionsare: Flow rate DNA captured Amount of DNA (ml/minute) % captured (mg)0.25 59 1.18 0.5 31.25 0.62 1 31.25 0.62

Third Apparatus Example

A third example of an apparatus according to the invention is shown byFIG. 14. This embodiment has all basic features described above for thefirst apparatus example, but comprises e.g. two pairs of poles 81, 82and 83, 84, each pair belonging to a respective electromagnet which isconnected to a respective electrical current source. These are e.g. ACcurrent sources and the magnetic fields created therewith are preferablyout of phase, the phase difference being e.g. of 90 degrees. Suchmagnetic fields cooperate to retain the magnetic particles withinflow-through cell 18 and to act on the retained magnetic particles insuch a way that they are even more homogeneously distributed in theinterior of flow-through cell 18.

FIG. 15 shows a cross-sectional view of the quadrupole configuration ofpoles shown by FIG. 14.

Other embodiments similar to the one shown by FIGS. 14 and 15 comprisemore than two pairs of poles and consequently more that twoelectromagnets, which receive electrical currents having phase delayswith respect to each other. Since the magnetic field generated has inthis case a spherical symmetry, such embodiments make it possible toobtain a better distribution of the retained magnetic particles withinthe flow-through cell, instead of a distribution of the retainedmagnetic particles limited to those contained within a cylindricalsegment of the flow-through cell, as is the case in the more simpleembodiments described with reference e.g. to FIGS. 1 to 7.

Fourth Apparatus Example

A fourth example of an apparatus according to the invention is describedhereinafter with reference to FIG. 16 and 17. This embodiment hasfeatures similar to those described above for the first apparatusexample, but comprises three poles 91, 92 and 93 which belong to anelectromagnet arrangement having a magnetic core 97 which has three armseach of which ends in one of the poles 91, 92 and 93. A flow-throughcell 98 is arranged in the air gap between poles 91, 92 and 93.

Pole 92 is symmetrically arranged with respect to poles 91 and 93. Inmore general terms, three or more poles are symmetrically arranged withrespect to each other.

Each of the three arms of magnetic core 97 is associated with arespective winding 94, 95 and 96 respectively. Each of these windings isconnected to a respective electrical current source (not shown in FIG.16). These may be e.g. AC current sources and the magnetic fieldscreated therewith may be out of phase, the phase difference being e.g.of 90 degrees. Such magnetic fields cooperate to retain the magneticparticles within flow-through cell 98 and to act on the retainedmagnetic particles in such a way that they are even more homogeneouslydistributed in the interior of flow-through cell 98.

FIG. 17 shows a perspective cross-sectional view of the three-poleconfiguration shown by FIG. 16.

The operation of the three-pole embodiment shown by FIGS. 16 and 17 ischaracterized in that by means of a suitable choice of the time variableelectrical currents applied to at least one of windings 94, 95 and 96respectively, the resulting variable magnetic field generated andapplied to the interior of the flow-through cell 98 has no zero value atany time and makes thereby possible to obtain a better distribution ofthe retained magnetic particles within the flow-through cell.

Embodiments of the Apparatuses Described Above with Reference to FIGS.1-17

Embodiments of the apparatuses described above with reference to FIGS.1-17 are characterized by the following features taken alone or incombination:

-   -   a) the width H of the outer surface of the tapered poles is        equal to the thickness of the air gap,    -   b) the depth h of the outer surface of the tapered poles is        substantially equal to the depth of the flow-through cell,    -   c) the distance l between the of the outer surfaces of two        adjacent tapered poles is larger than the width H of a tapered        pole,    -   d) the specific dimensions and the number of the tapered poles        are configured in correspondence with the amount and the desired        distribution of the magnetic particles to be retained within the        flow-through cell,    -   e) at least two poles are symmetrically arranged with respect to        each other,    -   f) at least two poles are used for generating a magnetic field        characterized by a predetermined time variation in amplitude and        polarity,    -   g) at least two poles are used for generating a magnetic field        characterized by a predetermined phase with respect to a given        reference, and/or    -   h) the apparatus comprises more than two poles and those poles        are used for generating a composite magnetic field having a time        variation in amplitude and polarity that is the result of the        superposition of phase and time variation in amplitude and        polarity of the magnetic fields generated by each pair of the        plurality of poles, and the composite magnetic field is        preferably suitable for retaining magnetic particles under a        flow-through condition and to cause a magnetic particle dynamic        behavior which leads to a substantially uniform distribution of        the magnetic particles over the cross-section of the        flow-through cell.

Example of a First Method According to the Invention

According to the invention a first method for retaining magneticparticles within a segment of a flow-through cell during flow of a fluidthrough the cell comprises e.g. the following steps:

-   -   (a) inserting a flow-through cell into an air gap of at least        two electromagnets which have pole tips each having an outer        surface that faces the air gap and a shape that enables the        generation of an magnetic field gradient in the interior of the        flow-through cell,    -   (b) introducing into a flow-through cell an amount of magnetic        particles to be retained within a segment of that cell,    -   (c) applying a magnetic field having an amplitude and polarity        that vary with time to the space within the cell by means of the        at least two electromagnetic poles in order to retain the        magnetic particles within a segment of that flow-through cell,        and    -   (d) causing a fluid carrying molecules or particles to be        captured by the magnetic particles to flow through the        flow-through cell, e.g. by pump means connected to the        flow-through cell.

In one embodiment of the above-mentioned method the magnetic fieldapplied not only retains, but also uniformly distributes the magneticparticles within a segment of the flow-through cell.

In another embodiment, the variation of the magnetic field with time isa time variation of the amplitude, polarity, frequency of the magneticfield or a combination thereof.

In a further embodiment, the variation of the magnetic field is obtainedby a superposition of several magnetic field components, and eachcomponent is generated by an electromagnet of a set of electromagnets.

In another embodiment, the structure formed by the retained magneticparticles covering the entire cross-section of the flow-through channelis defined by the configuration of the time-varied magnetic field, whichconfiguration is defined by the parameters characterizing the magneticfield, namely the variation with time of its amplitude, frequency andpolarity.

A method of the above-mentioned kind may be carried out with one of theabove described examples of an apparatus according to the invention.

The electromagnet, the flow-through cell, the magnetic particles, andthe size of the flow of liquid through the flow-through cell may be soconfigured and dimensioned that the magnetic particles retained withinthe flow-through cell are distributed substantially over the entirecross-section of the flow-through cell, the cross-section being normalto the flow direction. The magnetic particles retained preferably form asubstantially homogenous suspension contained within a narrow segment ofthe flow-through cell.

The magnetic field applied may be varied with time in such a way thatthe magnetic particles retained within the flow-through cell form adynamic and homogeneous suspension wherein the magnetic particles are inmovement within a narrow segment of the flow-through cell.

The black surfaces 41 in FIG. 3 schematically represents a segment offlow-through cell 18 wherein the magnetic particles retained arehomogeneously distributed either as a stationary array if a staticmagnetic field is applied or as a dynamic group of moving particles if avariable magnetic field is applied. In the latter case the apparatusaccording to the invention not only retains the magnetic particleswithin a segment of the flow-through cell, but also manipulates them bymoving the particles with respect to each other during the retentionstep. This manipulation improves the contacts and thereby theinteraction between the target particles and the magnetic particles andprovides thereby a highly desirable effect for the diagnostic assays.

As shown in FIG. 3 each of segments 41 extends between opposite poletips.

FIGS. 8 and 9 illustrate possible distributions of the magneticparticles retained within the flow-through cell depending from thecharacteristics of magnetic field applied and the amount and density ofthe magnetic particles available within the flow-through cell. Thedensity of the magnetic particles is their mass divided by the volumewherein they are distributed.

FIG. 8 shows a cross-sectional view of the distribution of the magneticparticles 42 within flow-through cell 18 positioned between poles 21 and22 of electromagnet 13 in FIG. 1 before a liquid flows throughflow-through cell 18 and in two possible situations:

-   -   when the magnetic particles are under gravity force alone (arrow        43 shows the sense of gravity force), that is when no magnetic        field is applied, or    -   when a static magnetic field is applied and the density of the        magnetic particles is lower that a certain limit value.

FIG. 9 shows a cross-sectional view of the distribution of the magneticparticles 42 retained within flow-through cell 18 positioned betweenpoles 21 and 22 of electromagnet 13 in FIG. 1 when an alternatingmagnetic field is applied according to the invention and even when arelatively low density of magnetic particles is used. As alreadymentioned above, in the latter case the magnetic particles retained havea dynamic behavior and in particular relative motion with respect toeach other. Under the conditions just described the magnetic particles42 are retained within flow-through cell even when a liquid carryingtarget particles flows through flow-through cell 18, provided that theintensity of the flow does not exceed a certain limit value.

FIG. 10 shows a diagram (flow of liquid in milliliter per minute vs.magnetic field in Tesla) illustrating the retention capability that canbe obtained with an apparatus according to the invention operating withan alternating magnetic field of 2 cycles per second and a flow-throughcell 18 having an internal diameter of 1.5 millimeter provided that asufficient amount of magnetic particles is used. For liquid flow havinga value higher than the values delimited by the inclined line in FIG. 10the flow is strong enough to overcome the forces which retain themagnetic particles within the flow-through cell, and when this happensthe flow takes these particles away from flow-through cell 18. Theinclined line in FIG. 10 is defined by a number of points represented byblack squares. As shown in FIG. 10 these points lie within a range ofvariation.

In order to attain one of the main aims of the invention, which is toretain within a flow-through cell magnetic particles distributed overits entire cross-section under a certain flow of liquid carrying targetparticles, the following guidelines should be duly considered:

In order to have a magnetic field gradient which is large enough overthe whole depth of the gap,

-   -   the depth of the air gap between opposite pole tips should not        be larger than 0.1 to 10 millimeter,    -   the width H (shown in FIG. 13) of each pole tip surface should        not exceed a certain value, H should have a size of a few        millimeters, e.g. between 0.1 and 3 millimeter, and    -   the density of particles, i.e. the mass of magnetic particles        available within the flow cell divided by the volume of the flow        cell, should be larger than a minimum value.

Such a minimum density value corresponds e.g. to a mass of magneticparticles of 2 milligrams for the example described with reference toFIG. 13. If the density of magnetic particles is lower than a minimumvalue, the magnetic particles are not able to get distributed over theentire cross-section. On the other hand there is also a maximum value ofthe density of magnetic particles to be observed. For instance, if amass of magnetic particles larger than e.g. 5 milligrams is used for theexample described with reference to FIG. 13, then a part of the magneticparticles cannot be retained by the magnetic forces and is carried awayby the liquid flowing through the flow-through cell.

The value of magnetic susceptibility (also called magnetic force) of themagnetic particles plays also an important role for the operation of anapparatus according to the invention. The above indicated aims of theinvention are for instance obtained with an alternating magnetic fieldwith an amplitude of 0.14 Tesla and with magnetic particles having asusceptibility of approximately 0.5 Newton per kilogram. If the lattersusceptibility and/or the magnetic field amplitude were reduced to lowervalues, at some point the desired effect of a distribution of themagnetic particles over the entire cross-section of the flow-throughcell would not be obtainable.

The size and the number of the magnetic particles can be varied over arelatively large range without affecting the desired operation of anapparatus according to the invention. A decrease of the size of themagnetic particles can be compensated by a corresponding increase intheir number and vice versa.

Fifth Apparatus Example

A very localized high magnetic field is necessary for manipulatingmagnetic particles. When a microchannel is used as flow-through cell,the magnetic field and the magnetic field gradient have to be localizedin a microscopic scale, which is not achievable using a large externalpermanent magnet or electromagnet. As described below, according to theinvention, a magnetic field having the above-mentioned properties may begenerated by means of microstructured magnetic material layers which arelocated near to the microchannel and which the magnetic flux generatedby an external magnet.

FIGS. 18 to 20 show various views of a fifth apparatus according to theinvention. This apparatus has a microchip like structure and is suitablefor retaining magnetic particles within a segment of a microchannelflow-through cell during flow of a fluid through the cell. As shown byFIG. 18 this apparatus comprises a first layer 101 of a non-magneticmaterial comprising a rectilinear microchannel 102 which has apredetermined depth and which is suitable for use as a flow-throughcell. Microchannel 102 is suitable for allowing flow of liquid and forreceiving an amount of magnetic particles to be retained within asegment of microchannel 102. First layer 101 has a first opening 105 anda second opening 106. These openings are located on opposite sides ofmicrochannel 102. Each of openings 105, 106 is adapted for receiving aferromagnetic material sheet 107 respectively 108 having a shape thatmatches the shape of the respective opening 105 respectively 106.

The apparatus shown by FIG. 18 further comprises a first ferromagneticmaterial sheet 107 and a second ferromagnetic material sheet 108 each ofwhich snuggly fits into a corresponding one of openings 105 and 106respectively and is suitable for use as an end part of anelectromagnetic circuit.

Sheets 107 and 108 have each an outer surface which faces microchannel102. As shown by FIG. 19, the latter outer surface comprises the outersurfaces of at least two cavities 111 and 112 and of a tapered end part113 which separates cavities 111 and 112 from each other. The cavitiesand the tapered end part of the first sheet 107 of ferromagneticmaterial are arranged substantially opposite to and symmetrically withrespect to the corresponding cavities and tapered end part of the secondsheet 108 of ferromagnetic material. As shown by FIGS. 18 and 19 each ofsheets 108 and 109 may have a plurality of cavities 111, 112 and aplurality of tapered end parts 113.

The apparatus shown by FIG. 18 further comprises a second layer 114 of anon-magnetic material which covers the first layer 101 as well as thefirst and a second ferromagnetic material sheets 107, 108 lodged inopenings 105, 106 of first layer 101 of a non-magnetic material.

In one embodiment the first and a second ferromagnetic material sheets107, 108 each have a thickness which is approximately equal to the depthof microchannel 102.

FIG. 20 shows a cross-sectional view of another embodiment of theapparatus shown by FIGS. 18 and 19. This embodiment further comprises anelectromagnet 121 which has magnetic pole ends 123 and 124. In thisembodiment, the second layer 114 has two openings 115, 116. Each of poleends 123, 124 extend through one of openings 115, 116. Pole end 123,pole end 124 are each in contact with one of ferromagnetic materialsheets 107, 108. In FIG. 20, the assembly 125 comprises the first layer101, the second layer 114 and the ferromagnetic material sheets 107 and108.

The width of each tapered end parts 113 may be equal to the thickness ofthe gap between the outer surfaces of the first and second ferromagneticmaterial sheets.

The depth of the tapered end parts 113 may be substantially equal to thedepth of microchannel 102.

The distance between two adjacent tapered end parts 113 may be largerthan the width of a tapered end part 113.

The specific dimensions and the number of the tapered end parts 113 maybe configured in correspondence with the amount and the desireddistribution of the magnetic particles to be retained withinmicrochannel 102.

The embodiment described above with reference to FIGS. 18 to 20 issuitable for retaining magnetic particles having a size that lies in thenanometer or micrometer range.

Such particles may be of the kind used for capturing target molecules ortarget particles carried by the liquid.

Example of a Second Method According to the Invention

According to the invention a second method for retaining magneticparticles within a segment of a microchannel used as a flow-through cellduring flow of a fluid through the microchannel comprises e.g. thefollowing steps:

-   -   (a) positioning a microchannel used as a flow-through cell        between ferromagnetic material sheets each having an outer        surface that faces the microchannel, that outer surface having a        shape that enables the generation of an magnetic field gradient        in the interior of the microchannel when a magnetic field is        applied by means of the ferromagnetic material sheets,    -   (b) introducing into the microchannel an amount of magnetic        particles to be retained within a segment of that microchannel,    -   (c) applying a magnetic field having an amplitude and polarity        that vary with time to the space within the microchannel by        means of the ferromagnetic material sheets in order to retain        the magnetic particles within a segment of the microchannel,    -   (d) causing a fluid carrying molecules or particles to be        captured by the magnetic particles to flow through the        microchannel.

In one embodiment the magnetic field not only retains, but alsouniformly distributes the magnetic particles within a segment of themicrochannel.

Apparatuses or a methods according to the invention are suitable for usein a life science field and in particular for in-vitro diagnosticsassays, therefore including applications for separation, concentration,purification, transport and analysis of analytes (e.g. nucleic acids)bound to a magnetic solid phase of a fluid contained in a reactioncuvette or in a fluid system (channel, flow-through cell, pipette, tip,reaction cuvette, etc.).

Although preferred embodiments of the invention have been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

1. An apparatus for retaining magnetic particles within a segment of aflow-through cell during flow of a fluid through said cell comprising(a) an electromagnet comprising a winding connectable to a currentsource, said electromagnet having at least two poles separated by an airgap which is much smaller than the overall dimensions of theelectromagnet,. said air gap lying between the outer surfaces of theends of said at least two poles, each of the latter outer surfacescomprising the outer surfaces of at least two cavities and of a taperedpole end part which separates said at least two cavities from eachother, the cavities and the tapered end part of one of the poles beingarranged substantially opposite to and symmetrically with respect to thecorresponding cavities and tapered end part of the other pole of said atleast two poles, the depth of the air gap thereby varying at least alonga first direction, said depth being measured along a second directionnormal to said first direction, and said gap having at least a firstsymmetry axis which extends along said first direction; and (b) aflow-through cell which is suitable for receiving an amount of magneticparticles to be retained within a segment of the flow-through cell andto allow flow of a liquid through the flow-through cell, and a portionof said flow-through cell being inserted in said air gap in such a waythat at least one area of the outer surface of each of said tapered poleparts is in contact with or close to the outer surface of a wall of saidflow-through cell and the length axis of said flow-through cell portionextends along said first direction.
 2. An apparatus according to claim1, wherein the size of the magnetic particles is less than or equal toabout 5 μm.
 3. An apparatus according to claim 1, wherein the magneticparticles are effective to capture target molecules or target particlespresent in said liquid.
 4. An apparatus according to claim 1, whereinthe air gap has an average thickness between 0.1 and 10 millimeters. 5.An apparatus according to claim 1, wherein the width of the of the outersurface of the tapered poles is equal to the thickness of the air gap.6. An apparatus according to claim 1, wherein the depth of the outersurface of the tapered poles is substantially equal to the depth of theflow-through cell.
 7. An apparatus according to claim 1, wherein thedistance between the outer surfaces of two adjacent tapered poles isgreater than the width of a tapered pole.
 8. An apparatus according toclaim 1, wherein the specific dimensions and the number of the taperedpoles are configured in correspondence with the amount and the desireddistribution of the magnetic particles to be retained within theflow-through cell.
 9. An apparatus according to claim 1, wherein said atleast two poles are symmetrically arranged with respect to each other.10. An apparatus according to claim 1, wherein said at least two polesare used for generating a magnetic field characterized by apredetermined time variation in amplitude and polarity.
 11. An apparatusaccording to claim 1, wherein said at least two poles are used forgenerating a magnetic field characterized by a predetermined phase. 12.An apparatus according to claim 1, said apparatus comprising more thantwo poles and said poles being effective for generating a compositemagnetic field having a time variation in amplitude and polarity that isthe result of the superposition of phase and time variation in amplitudeand polarity of the magnetic fields generated by each pair of saidplurality of poles.
 13. An apparatus according to claim 12, wherein saidcomposite magnetic field is suitable for retaining magnetic particlesunder a flow-through condition and with a substantially uniformdistribution of the magnetic particles over the cross-section of theflow-through cell.
 14. An apparatus according to claim 1, wherein theelectrical current source is a source adapted to provide a current whichis variable with time.
 15. An apparatus according to claim 14, whereinthe electrical current source is an alternating current source.
 16. Anapparatus according to claim 15, wherein the alternating current sourceis adapted to supply a current having a selectable frequency comprisedbetween 0.001 cycle per second and 100 kilocycles per second.
 17. Anapparatus according to claim 14, wherein the electric current source isan switchable DC current source.
 18. An apparatus according to claim 1,wherein the electric current source is a DC current source.
 19. Anapparatus according to claim 1, wherein the cavities and tapered poleend parts form a corrugated surface.
 20. An apparatus according to claim1, wherein each of said tapered pole end parts has a three-dimensionalshape.
 21. An apparatus according to claim 19, wherein said corrugatedsurface has a thickness comprised between 0.1 and 10 millimeters.
 22. Anapparatus according to claim 1, wherein said at least two cavities aregrooves or channels parallel to each other, the length axis of each ofsaid grooves or channels extending along a third direction which isnormal to a plane defined by a first axis in said first direction and asecond axis in said second direction.
 23. An apparatus according toclaim 22, wherein each of said grooves or channels has a cross-sectionhaving the shape of a half circle.
 24. An apparatus according to claim22, wherein each of said grooves or channels have a cross-section havingan undulated shape or a sawtooth shape.
 25. An apparatus according toclaim 1, wherein said at least two cavities and said tapered pole endparts are formed by the intersection of a first set of grooves orchannels parallel to each other, the length axis of each of said groovesor channels extending along a third direction which is normal to a planedefined by a first axis in said first direction and a second axis insaid second direction, with a second set of grooves or channels parallelto each other, the length axis of each of said grooves or channelsextending along said first direction.
 26. An apparatus according toclaim 25, wherein each of said grooves or channels of said first set ofgrooves or channels and of said second set of grooves or channels has across-section having the shape of a half circle.
 27. An apparatusaccording to claim 25, wherein each of said grooves or channels of saidfirst set of grooves or channels and of said second set of grooves orchannels has a cross-section having a wave-like or sawtooth shape. 28.An apparatus according to claim 25, wherein each of said tapered poleend parts has a flat outer surface facing said air gap.
 29. An apparatusaccording to claim 25, wherein each of said tapered pole end parts endsin a ridge.
 30. An apparatus according to claim 1, wherein each of saidtapered pole end parts is made of a ferromagnetic material.
 31. Anapparatus according to claim 30, wherein said material is a ferrite. 32.An apparatus according to claim 1, wherein said cavities are made bypowder blasting.
 33. A method for capturing target molecules or targetparticles carried by a liquid comprising: (a) forming a structure ofmagnetic particles distributed over a cross-section of a flow-throughcell, said structure being formed by (1) inserting a flow-through cellinto an air gap of at least two electromagnets which have pole tipshaving each an outer surface that faces said air gap and a shape thatenables the generation of an magnetic field gradient in the interior ofthe flow-through cell, (2) introducing into said flow-through cell anamount of magnetic particles to be retained within a segment of saidflow-through cell, (3) applying a magnetic field having an amplitude andpolarity that vary with time to the space within said cell by means ofsaid at least two electromagnets in order to retain said magneticparticles within a segment of said flow-through cell, and (b) causingsaid liquid carrying target molecules or target particles to flowthrough said structure of magnetic particles retained within saidsegment of said flow-through cell.
 34. A method according to claim 33,wherein said magnetic field uniformly distributes said magneticparticles within a segment of the flow-through cell.
 35. A methodaccording to claim 33, wherein said outer surface of said pole tips is acorrugated surface.
 36. A method according to claim 33, wherein theelectromagnet, the flow-through cell, the magnetic particles, and thesize of the flow of liquid through the flow-through cell are soconfigured and dimensioned that the magnetic particles retained aredistributed substantially evenly over the entire cross-section of theflow-through cell, said cross-section being normal to the flowdirection.
 37. A method according to claim 36, wherein the magneticparticles retained form a substantially homogenous suspension containedwithin a segment of the flow-through cell which is substantially normalto the flow direction.
 38. A method the according to claim 37, whereinthe magnetic field applied is varied with time in order to cause theretained magnetic particles to form a dynamic and homogeneous suspensionwherein the magnetic particles are in movement within said segment. 39.A method according to claim 38, wherein the variation of the magneticfield with time is a time variation of at least one of the amplitude,polarity, and frequency of the said magnetic field.
 40. A methodaccording to claim 38, wherein the variation of the magnetic field isobtained by a superposition of several magnetic field components, eachcomponent being generated by one electromagnet of a set ofelectromagnets.
 41. A method according to claim 38, wherein thestructure formed by the retained magnetic particles covering the entirecross-section of the flow-through channel is defined by theconfiguration of the time-varied magnetic field, which configuration isdefined by variations in one or more of the amplitude, frequency andpolarity of the magnetic field.
 42. A method for maximizing the surfacesof magnetic particles that are contacted by liquid which carries targetmolecules or target particles and flows through a flow-through cellcomprising: (a) forming a structure of magnetic particles distributedover a cross-section of said flow-through cell, said structure beingformed by (1) inserting a flow-through cell into an air gap of at leasttwo electromagnets which have pole tips having each an outer surfacethat faces said air gap and a shape that enables the generation of anmagnetic field gradient in the interior of the flow-through cell, (2)introducing into said flow-through cell an amount of magnetic particlesto be retained within a segment of said flow-through cell, (3) applyinga magnetic field having an amplitude and polarity that vary with time tothe space within said cell by means of said at least two electromagnetsin order to retain said magnetic particles within a segment of saidflow-through cell, and (b) causing said liquid carrying target moleculesor target particles to flow through said structure of magnetic particlesretained within said segment of said flow-through cell.
 43. An apparatusfor retaining magnetic particles within a segment of a flow-through cellduring flow of a fluid through said cell comprising (a) a first layer ofa non-magnetic material comprising a rectilinear microchannel which hasa predetermined depth and which is suitable for use as a flow-throughcell, said microchannel being suitable for allowing flow of liquid andfor receiving an amount of magnetic particles to be retained within asegment of said microchannel, said first layer having a first openingand a second opening located on opposite sides of said microchannel,each of said openings being adapted for receiving each a ferromagneticmaterial sheet having a shape that matches the shape of the respectiveopening, (b) a first ferromagnetic material sheet and a secondferromagnetic material sheet each of which snuggly fits into arespective one of said openings of said first layer and is suitable foruse as an end part of an electromagnetic circuit, each of said sheetshaving each an outer surface which faces said microchannel, said outersurface comprising the outer surfaces of at least two cavities and of atapered end part which separates said at least two cavities from eachother, the cavities and the tapered part of the first sheet offerromagnetic material being arranged substantially opposite to andsymmetrically with respect to the corresponding cavities and tapered endpart of the second sheet of ferromagnetic material, and (c) a secondlayer of a non-magnetic material which covers said first layer and thefirst and a second ferromagnetic material sheets lodged in said openingsof said first layer of a non-magnetic material.
 44. An apparatusaccording to claim 43, wherein the first and second ferromagneticmaterial sheets have each a thickness which is approximately equal tothe depth of said microchannel.
 45. An apparatus according to claim 43,which further comprises an electromagnet having magnetic pole ends andwherein said second layer has two openings through which said pole endsextend, said pole ends being in contact with said first and secondferromagnetic material sheets.
 46. An apparatus according to claim 43,wherein the size of the magnetic particles is less that or equal toabout 5 μm.
 47. An apparatus according to claim 43, wherein the magneticparticles are effective to capture target particles present in saidliquid.
 48. An apparatus according to claim 43, wherein the width of thetapered end parts is equal to the thickness of the gap between saidouter surfaces of said first and second ferromagnetic material sheets.49. An apparatus according to claim 43, wherein the depth of saidtapered end parts is substantially equal to the depth of saidmicrochannel.
 50. An apparatus according to claim 43, wherein thedistance between two adjacent tapered end parts is greater than thewidth of a tapered end part.
 51. An apparatus according to any of claim43, wherein the microchannel has an average thickness, which liesbetween 10 micrometers and 1 millimeter.
 52. An apparatus according toclaim 43, wherein the specific dimensions and the number of the taperedend parts are configured in correspondence with the amount and thedesired distribution of the magnetic particles to be retained withinsaid microchannel.
 53. A method for retaining magnetic particles withina segment of a microchannel used as a flow-through cell during flow of afluid through said microchannel comprising (a) positioning amicrochannel used as a flow-through cell between ferromagnetic materialsheets suitable for collecting a magnetic field generated by anelectromagnet, each of said sheets having an outer surface that facessaid microchannel, said outer surface having a shape that enables thegeneration of an magnetic field gradient in the interior of themicrochannel when a magnetic field is applied to said microchannel bymeans of said ferromagnetic material sheets, (b) introducing into saidmicrochannel an amount of magnetic particles to be retained within asegment of that microchannel, (c) applying a magnetic field having anamplitude and polarity that vary with time to the space within saidmicrochannel by means of said ferromagnetic material sheets in order toretain said magnetic particles within a segment of the microchannel, (d)causing a fluid carrying molecules or particles to be captured by themagnetic particles to flow through the microchannel.